Our multiplex imaging technique previously allowed us to fully characterize all the migratory and resident dendritic cell subpopulations in skin draining lymph nodes of mice and to reveal the complex and heterogeneous, but non-random distribution of these various dendritic cell subsets. We call this entire set of analytic tools Histo-cytometry for its similarity to flow cytometric analysis. This past year, we applied this method to a quantitative analysis of antigen delivery in lymph nodes, showing that even for small materials, penetration through conduits to deeper regions is quite limited. We found that DC position played a crucial role in the quantity of antigen acquired and that small changes in vaccine dose could selectively limit CD8 T cell activation, with important implications for design of vaccines geared toward elicitation of cell mediated immune responses. Our earlier studies showed the dramatically asymmetric distribution of major DC subsets with the lymph node architecture, with so-called cDC1s mainly positioned in the center of the T cell paracortical region and cDC2s in more peripheral locations, including the subcapsular sinus floor. Because cDC1 and cDC2 preferentially present antigens via MHC class I and MHC class II molecules respectively, they would be expected to interact with and activate CD8 and CD4 T cell preferentially. Given the critical role of spatial organization in the efficient operation of the adaptive immune system, we considered the possibility that there might be asymmetries in the distributions of these two subsets that better matched the asymmetry of the cDC1 / cDC2 distributions to permit more efficient and rapid association of the relevant T cells with their matched antigen presenting cells. Using Histo-cytometry, we indeed discovered that there is a biased distribution of CD4 T cells within nave lymph nodes (See also ZIA AI000758-21 LSB). Based on gene transcription data, the oxy-sterol receptor Ebi2, which had already been shown to control peripheralization of lymphocytes within lymph nodes, was a prime candidate for the molecule controlling this positioning. Ebi2 deficient mice showed a loss of the asymmetric distribution of CD4 T cells, which was also seen after transfer of labelled wild type and Ebi2 KO T cells into wild type hosts. Functional relevance of this control of CD4 T cell positioning was seen following infection with helminths, with a delay in expulsion and prolonged egg development and also in a liver stage malaria vaccine model where protection is dependent on CD8 Trm in the liver. Loss of Ebi2 polarization resulted is loss of nearly all the protective capacity of the primed memory CD8 T cells. Together, these data emphasize the critical role played by spatial topography within secondary lymphoid tissues and in particular, the crucial role of spatial matching between T cell subsets and DC subsets in ensuring an efficient response to infection. In a new application of Histo-cytometry, we developed methods for combining cell phenotyping with analysis of phospho-proteins, especially pSTAT molecules. We previously reported on the discovery of small clusters of pSTAT5 Tregs surrounding activated conventional T cells and the role of Treg TCR signaling and IL-2 stealing in functional control of self-reactive Tv cell responses. We have now extended the use of pSTAT imaging to the gut. Many of the cytokines involved in communication between lymphocytes and epithelial cells in the small bowel signal via STAT3. We employed Histo-cytometry involving staining for epithelial cells, CD3, RORgt (a key transcription factor for Th17 T cells and for ILC3s), and pSTAT3, among a number of target molecules examined, and compared the ileal regions of wild-type (WT) and RAG KO mice. Surprisingly, nearly all epithelial cells in RAG KO animals showed strong pSTAT3 staining long with pSTAT3+ CD3- RORgt lymphocytes (ILC3s) in cryptopatches of that tissue. Further analysis revealed that the pSTAT3 signal was dependent on the microbiome and in particular, segmented filamentous bacteria (SFB). Additional studies showed that transfer of polyclonal CD4+ T cells into RAG KO mice eliminated the pSTAT3 signal indicating that effector functions not dependent on cytokines eliciting a pSTAT3 response were inhibiting the ILC3 response. This could be ascribed to Th17 reduction in SFB colonization that depressed IL-23 production, along with Treg action of CCR2+ myeloid cells that also limited IL-23 generation, in turn preventing production of IL-22 that was responsible for the pSTAT3 signaling in the epithelial cells. The physiological relevance of this trans-dominant role of adaptive T cells was revealed in studies showing that persistent high IL-22 secretion by ILC3s led to loss of lipid transporter expression by the epithelial cells, low serum FFA and triglycerides, and low adiposity, a pathologic state prevented by the suppression of ILC3s activity by the CD4 T cells. Thus, these studies revealed that adaptive CD4 T cells and ILC3s have quite distinct roles in setting up a noninflammatory commensal microbiota and in maintaining metabolic homeostasis. To extend our Histo-cytometry we have moved in two directions. First, we have developed a method (IBEX) that permits performing rapid iterative 6-10 color fluorescent immunohistochemistry on lymphoid and other tissues to attain images with >30 markers analyzed and to computationally analyze the data to assign multiple stains to specific cells. To examine tissue volumes rather than sections, we have developed a novel tissue clearing method called Ce3D. This is simpler than existing methods, produces superior transparency, preserves fluorescent protein signals, and also allows multiplex staining with diverse fluorochrome labeled antibodies. It promises to provide an unprecedented level of insight into cell organization, activity, and function in tissue samples, especially biopsies from patients, for example, those undergoing checkpoint blockade therapy in cancer. We are now extending its utility in a collaboration with Dr. Garry Nolan of Stanford University. Using a method his group has developed called CODEX that involves detection of oligo-labeled antibodies by short oligonucleotides coupled to fluorescent molecules, we have been able to extend Ce3D from a single cycle with 6-8 antibodies to an iterative method that can detect dozens of targets. We have also made progress in combining this new method with RNA-Fish so that data from single cell or bulk RNA seq can be used in conjunction with mAb staining for identification of cells and their state. To extend our understanding of how immune cells navigate in 3D tissue spaces, we employed micro fabricated devices that permit precise control of chemoattractant gradient steepness and magnitude. Using these 3D systems, our findings suggest that temporal rather than spatial sensing plays a crucial role in persistent directional migration of various myeloid cell types to what are called guidance chemokines, and that the difference can be attributed to distinct ways in which negative feedback regulation operates with respect to the different classes of chemoattractants. We have developed a novel method for imaging the peritoneal wall, allowing us to study dynamically and in depth the roles of neutrophils, inflammatory monocytes/macrophages, and tissue resident macrophages in response to tumors, pathogens, and sterile injury. Remarkably, embryologically-derived fixed tissue macrophages responded in a 2-phase manner to single cell sized sterile damage with a cloaking behavior that shields the damaged area from neutrophils and prevents the latter from swarming and increasing the lesion size. We now have evidence that this process is key to maintenance of tissue homeostasis in the face of episodic cell death in various tissues.